Wang et al. Cell Death and Disease (2021) 12:387 https://doi.org/10.1038/s41419-021-03679-8 Cell Death & Disease ARTICLE Open Access ATF3 deficiency impairs the proliferative–secretory phase transition and decidualization in RIF patients Zhilong Wang 1,YangLiu1, Jingyu Liu1,NaKong1,YueJiang1,RuiweiJiang 1,XinZhen1, Jidong Zhou1, Chaojun Li 2, Haixiang Sun 1 and Guijun Yan 1 Abstract Decidualization is a complex process involving cellular proliferation and differentiation of the endometrial stroma and is required to establish and support pregnancy. Dysregulated decidualization has been reported to be a critical cause of recurrent implantation failure (RIF). In this study, we found that Activating transcription factor 3 (ATF3) expression was significantly downregulated in the endometrium of RIF patients. Knockdown of ATF3 in human endometrium stromal cells (hESCs) hampers decidualization, while overexpression could trigger the expression of decidual marker genes, and ameliorate the decidualization of hESCs from RIF patients. Mechanistically, ATF3 promotes decidualization by upregulating FOXO1 via suppressing miR-135b expression. In addition, the endometrium of RIF patients was hyperproliferative, while overexpression of ATF3 inhibited the proliferation of hESCs through CDKN1A. These data demonstrate the critical roles of endometrial ATF3 in regulating decidualization and proliferation, and dysregulation of ATF3 in the endometrium may be a novel cause of RIF and therefore represent a potential therapeutic target for RIF. Introduction 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; implantation failure (RIF) is one of the major causes of Reciprocal intimate cross-talk between the activated infertility in ART programs, and these pregnancy failures blastocyst and the receptive uterus is essential for suc- are believed to be mainly due to defects in early pregnancy cessful implantation, and therefore, for pregnancy out- events, including implantation and decidualization4. come1. The desire to have children is powerful and In recent years, it has become apparent that endometrial widespread, however, about 15% of couples are childless factors have an important role in implantation5,6. The because of infertility, which is a worldwide social and human endometrial cycle can be divided into 2 phases: the economic concern2. Currently, due to impressive advan- proliferative phase, which is marked by the active growth ces in assisted reproductive technology (ART) and, in of stromal, epithelial, and vascular cells, and the secretory particular, the advent of more effective embryo selection phase, during which the endometrium undergoes decid- and cryopreservation, many underlying causes of human ualization to prepare for implantation7,8. The proper infertility have been overcome; however, there are still transition between the proliferative and secretory phases many patients who continue to experience ART failure is a prerequisite for the establishment of the WOI and the after serial IVF attempts3, probably owing to embryos subsequent pregnancy. Decidualization deficiency or being transferred into a nonreceptive uterus. Recurrent abnormality may be one of the major maternal causes of RIF9,10. Besides, the receptivity window of RIF patients was delayed by 1–3 days11,12, suggesting that the acqui- Correspondence: Haixiang Sun ([email protected])or sition of the endometrial receptivity is slower in RIF Guijun Yan ([email protected]) 1Reproductive Medicine Center, The Affiliated Drum Tower Hospital of Nanjing patients. Nevertheless, the critical underlying molecular University Medical School, 210008 Nanjing, People’s Republic of China mechanisms remain largely unknown. 2 ’ Nanjing University Medical School, 210008 Nanjing, People s Republic of Activating transcription factor 3 (ATF3) belongs to the China These authors contributed: Zhilong Wang, Yang Liu, Jingyu Liu ATF/CREB transcription factor family, which binds to the Edited by Y. Shi © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2021) 12:387 Page 2 of 15 consensus ATF/cAMP response element in numerous phase than in the proliferative phase of the endometrium, promoters13,14. It can also regulate various biological especially in the endometrial stromal cells (Fig. 2A, B). We functions independent of its transcriptional activity15,16. also analyzed the relative expression of temporal ATF3 ATF3 acts as a hub of the cellular adaptive-response expression across the cycle from a public dataset (Fig. 2C). network and has vital roles in modulating metabolism, Additionally, a recent single-cell RNA-seq analysis sug- immunity, the cell cycle, and differentiation. Distinct from gests that ATF3 may be a probable driver for decid- other ATF/CREB proteins, ATF3 expression is rapidly ualization20, and using this data we found that ATF3 is induced by a wide range of extracellular signals, such as elevated in the stromal cells since the early-secretory nutrient deprivation, endoplasmic reticulum stress, cyto- phase (Fig. 2D). Besides, the expression of ATF3 in all – kines, chemokines, and hormones17 19. However, little is cells of the endometrium is upregulated in the mid- known about whether uterine ATF3 has any role in secretory compared with the early-secretory phase (Fig. decidualization, the proliferative–secretory phase transi- 2E). tion of the menstrual cycle, and the pathological rela- We also examined the levels of ATF3 and other mem- tionship with RIF. In this study, we describe a novel bers of the ATF/CREB family in hESCs treated with 8-Br- mechanism of dysregulation of ATF3 in the endometrium cAMP plus MPA for 0.5–16 h using qPCR. The results from RIF patients hampers decidualization that may be a reveal that ATF3 was rapidly elevated by ~40-fold at ~1 h new cause of RIF. in hESCs (Fig. 2F), while other ATFs showed no sig- nificant change (Supplemental Fig. S2). Western blot Results analysis also confirmed that the expression of ATF3 was ATF3 is deficient in the endometrium of RIF patients increased after in vitro decidualization (Fig. 2G). Gene set In our previous study, we demonstrated that the enrichment analysis (GSEA) results showed that the endometrium of RIF patients displays a distinct tran- ATF3 target term was significantly upregulated during scriptional profile and suggests that dysregulation of decidualization (NES = 1.436, P < 0.01) (Fig. 2H). The decidualization may be a cause of implantation failure10. results indicate that decidualization stimuli could upre- Through RNA-seq analysis, we found that ATF3 expres- gulate the expression of ATF3 in the stroma of the sion was significantly downregulated in the endometrium endometrium. of RIF women (Fig. 1A, B), while other members of ATF/ CREB did not change significantly (Supplemental Fig. S1). Lack of ATF3 impairs decidualization qPCR was then used to validate that in the mid-secretory Various functions have been ascribed to decidual pro- phase, subjects with RIF had a 0.5-fold lower ATF3 lactin (PRL) which is now established as a widely used expression level than controls (Fig. 1C), consistent with marker to assess the differentiation status of hESCs in the western blot results (Fig. 1D, E). As shown in Fig. 1F, culture. As shown in Supplemental Fig. S3A, the expres- G, the expression of ATF3 in the endometrial stromal sion of ATF3 showed a positive relationship with PRL in cells from RIF patients was significantly decreased, as the endometrium. When hESCs were transfected with determined by immunohistochemical staining. Through small interfering RNA specific for ATF3 for 48 h followed analysis of publicly accessible data banks, we found that by treatment with 8-Br-cAMP and MPA for 3 days, the the placenta exhibits a high level of ATF3 expression, expression and secretion of PRL were substantially whereas its level ranges from medium to high in the decreased (Fig. 2I, J). We further examined the effect of uterus and endometrium, indicating an important role for ATF3 on the reorganization of the cytoskeleton, which ATF3 in human endometrial cycling (Supplemental Fig. reflects a morphological marker of decidualization21.As S1). We, therefore, wondered whether an element of the shown in Fig. 2K, decidualized hESCs displayed polygonal infertility phenotype could be caused by an alteration in cell morphologies with a random distribution of F-actin the expression of ATF3. In addition, we found that the filaments. When knockdown of endogenous ATF3, stro- expression of the decidualization marker PRL was mal cells maintained a fibroblast-like phenotype under decreased in the endometrium from RIF patients (Fig. decidualization stimuli.